Row-independent oligonucleotide synthesis

ABSTRACT

Apparatuses and a method for plate-based oligonucleotide synthesis are disclosed. In one example, an apparatus used in oligonucleotide synthesis includes a machined block to receive a commercially-available synthesis plate. A keeper is used to apply pressure to the commercially-available synthesis plate, and a sealing element is used to seal the commercially-available synthesis plate to the machined block. Other methods and apparatuses are disclosed.

CLAIM OF PRIORITY

This patent application claims priority to and is a continuation of U.S.patent application Ser. No. 17/250,642, filed Feb. 15, 2021, which willissue as U.S. Pat. No. 11,596,919 on Mar. 7, 2023, which, pursuant to 35U.S.C. § 371, is a U.S. National Phase application of and claimspriority to PCT/US2019/046802, now WO2020037194, filed Aug. 16, 2019,which claims priority pursuant to 35 U.S.C. § 119(e) to U.S. ProvisionalApplication Ser. No. 62/719,487, filed Aug. 17, 2018. The text andcontents of each of these patent applications are hereby incorporatedinto this application by reference as though fully set forth herein.

TECHNOLOGY FIELD

The disclosed subject matter is generally related to the field ofbiotechnology. More specifically, the disclosed subject matter isrelated to the de novo synthesis of DNA, RNA, synthons, and fullgenes—frequently generically referred to as oligonucleotide synthesis.

BACKGROUND

Since the release of the seminal paper on tRNA synthesis in 1972 by H.G. Khorana et al., the field of gene synthesis has experienced steadygrowth. With its use in generating novel therapeutics and biomaterials,academic and industrial researchers frequently require more exogenousDNA sequences than a standard laboratory can produce.

To fill this need, automated oligonucleotide synthesis systems have beendeveloped to generate oligonucleotides in hours, in quantities andvarieties that a single laboratory technician would have otherwiseneeded weeks or months to complete. As the demand for syntheticoligonucleotides increases, these high-throughput systems mustexperience continual refinement to meet the needs of the marketplace.

The information described in this section is provided to offer theskilled artisan a context for the following disclosed subject matter andshould not be considered as admitted prior art.

SUMMARY

Devices, mechanisms, and design elements are disclosed herein thatreduce reagent consumption, increase throughput, and shorten cycle timeson an oligonucleotide synthesis apparatus. In an embodiment, a mechanismfor these improvements includes, in various embodiments, a machinedblock that can receive commercially-available synthesis plates andsynthesize unique genetic material in each well, while allowingself-contained rows of each of the plates to retain full autonomy withrespect to one another. This autonomy not only increases the versatilityof the plates, but also allows a user to conduct synthesis in acontinuum or gradient, thereby decreasing cycle times. Variousembodiments presented herein offer an end user processes for generatingoligonucleotides at a significantly reduced cost with significantlyhigher production rates.

In an embodiment, the disclosed subject matter includes an apparatusused for oligonucleotide synthesis. The apparatus includes a machinedblock configured to receive a commercially-available synthesis plate, akeeper to apply pressure to the commercially-available synthesis plate,and a sealing element to seal the commercially-available synthesis plateto the machined block.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a high-level, exemplary embodiment of a row-independentoligonucleotide (RIOS) system;

FIG. 2A shows an exemplary embodiment of a broken-out, disassembleddraining apparatus comprising a drain block, a sealing element, asynthesis plate, and a keeper in accordance with various embodiments ofthe disclosed subject matter;

FIG. 2B shows a top view of the synthesis plate of FIG. 2A;

FIG. 2C shows a side view of the synthesis plate of FIG. 2A;

FIG. 2D shows an enlarged view of waste tips of the synthesis plate ofFIG. 2A;

FIG. 3 shows an exemplary embodiment of an assembled draining apparatusin accordance with various embodiments of the disclosed subject matter;

FIG. 4 shows an exemplary embodiment of an expanded apparatus inaccordance with various embodiments of the disclosed subject matter;

FIG. 5 an exemplary embodiment of a drain-hole plate in accordance withvarious embodiments of the disclosed subject matter;

FIG. 6 an exemplary embodiment of a column-based drain block inaccordance with various embodiments of the disclosed subject matter;

FIG. 7 shows an exemplary embodiment of a gas/fluid pathway for a RIOSsystem under negative pressure in accordance with various embodiments ofthe disclosed subject matter;

FIG. 8 shows an exemplary embodiment of a gas/fluid pathway for a RIOSsystem under a hybridization of positive pressure and negative pressurein accordance with various embodiments of the disclosed subject matter;

FIG. 9 shows an exemplary embodiment of a dispense-tip assembly inaccordance with various embodiments of the disclosed subject matter;

FIG. 10 shows an exemplary embodiment of a lid housing forvalve-adjacent reagent bottles in accordance with various embodiments ofthe disclosed subject matter;

FIG. 11 shows an exemplary embodiment of a reverse-flush pathway inaccordance with various embodiments of the disclosed subject matter; and

FIG. 12 shows an exemplary embodiment of a calibration apparatus thatmay be used with various ones of the embodiments disclosed herein.

A person of ordinary skill in the art will recognize that variousdimensions and other units provided herein, including those dimensionsand other units provided in the appended figures, are given merely toprovide a context in which the disclosed subject matter may readily beunderstood. However, the dimensions and other units can be varied asneeded. Therefore, the dimensions and other physical units should not beconsidered as being limiting; the skilled artisan, upon reading andunderstanding the disclosure provided herein, will recognize how tomodify various ones of the dimensions and other units as needed for agiven application.

DETAILED DESCRIPTION

Despite the potential for variability and speed offered in plate-basedcreation of DNA, conventional synthesis has not taken this approach.Instead, individual columns have been the tool-of-choice forsynthesizing oligonucleotides. The use of individual columns has beenused for a variety of reasons, including:

-   -   1) The excess reagents required for plate-based synthesis        compromise the cost benefits. Drainage of a plate typically        requires that the entirety of the plate be drained at once,        meaning that to achieve full expulsion of used material (e.g.,        waste), a plate's wells must all be filled evenly so as to        achieve even drainage. In practice, this requirement for even        filling within the prior art plays out as a user having to fill        the unused wells of a plate with reagent only so that the used        wells may conduct a quality synthesis. The reagents used to fill        the excess wells in this process are therefore often wasted;    -   2) The time spent beginning synthesis on the first well and        ending on the last well has economic consequences as well as        chemical consequences. If a plate is conducting synthesis on all        wells, the time after the first well has completed synthesis and        is not draining is effectively time wasted. Whereas a synthesis        run in singular columns may have the benefit of continuing its        own synthesis despite an adjacent column's status, a plate's        wells are dependent on each other in that the first well cannot        drain until the last well can drain. Furthermore, the reagents        used in oligonucleotide synthesis are corrosive enough to        degrade a nascent DNA strand itself if left for too long. The        excessive wait times are often exploited by the more aggressive        chemicals involved, leading to a decline in the final yield of a        plate's synthesis; and    -   3) To mitigate the time constraints and other issues outlined        above, attempts have been made to provide valves for every        position on the plate so that each may start and finish        synchronously. Although this technique has proven to increase        oligonucleotide yields, the costs involved to include this        increased number of valves prices the machines out of the        budgets of most laboratories. In cases where labs can afford        these machines, the physical space required to accommodate such        a machine often does not justify the system's presence.

The apparatus described herein facilitates improvements in plate-basedDNA synthesis by resolving at least the issues noted above. It should benoted that the various embodiments disclosed herein will use an exampleintended for use with a 384-well plate. However, upon reading andunderstanding the disclosed subject matter, a person of ordinary skillin the art with recognize that the disclosed subject matter may beexpanded or scaled-down to seat plates of any size of orientation,though typically in, for example, configurations of 96, 384, or 1536wells as described herein merely for ease in understanding the variousembodiments described.

When inspecting the issues surrounding DNA synthesis in plates, a personof ordinary skill in the art will recognize that many or all problemsarise from one crucial flaw of prior art systems: DNA synthesis machinestreating a synthesis plate as a single vehicle for DNA synthesis.Instead, the DNA synthesis machine should have an ability to treat thesynthesis plate as though each row in a plurality of rows and/or wellcontained within the synthesis plate is independent of the other rows orwells. An immediate problem in the prior art designs lies in thepressure requirements needed to operate one well or row and how the useris to implement that pressure without simultaneously affecting the otherwells and rows. Also, to accomplish the various prior artimplementations with consideration of susceptibility of adjacent ones ofthe rows regarding contamination to and from one another.

Various embodiments of the apparatuses and methods disclosed hereinoffer solutions in the form of, for example, an air-tight seal betweenrows and/or wells of one or more synthesis plates as described in detailbelow.

For example, FIG. 1 shows a high-level, exemplary embodiment of arow-independent oligonucleotide (RIOS) system 100. Because of themultiplicity of chemicals used in a reaction cycle of DNA, reagents areused and then allowed to pass through so that new reagents can be addedto continue the synthesis cycle. To accomplish the treatment of thereagents, a synthesis plate 101 is inserted into a chamber 103 as shownin FIG. 1 . The chamber 103 fills with inert gas from a pressurizedgas-source 105 to displace any oxygen that would not be conducive to DNAsynthesis. In embodiments, the pressurized gas-source 105 provides fromabout 5 kPa to about 100 kPa of pressure to the chamber 103. Once thechamber 103 is filled with inert gas, individual wells 115 within thesynthesis plate 101 begin to receive reagents for synthesis.

The reagents are dispensed from, for example, a ceiling 117 of thechamber 103 through a set of solenoid valves 107. The skilled artisanwill recognize that other types of valves, known in the art, may beutilized as well. In one embodiment, the set of solenoid valves 107 isstationary. Consequently, the synthesis plate 101 moves underneath theset of solenoid valves 107 to ensure that the dispensed reagents aredelivered to the correct wells within the synthesis plate 101. Inanother embodiment, the synthesis plate 101 is stationary and the set ofsolenoid valves 107 may be moved with reference to the synthesis plate101. In still other embodiments, each of the synthesis plate 101 and theset of solenoid valves 107 can be moved relative to one another. Forease of understanding the various embodiments of the disclosed subjectmatter, the remainder of the disclosure is based on an assumption thatthe synthesis plate 101 moves underneath a stationary version of the setof solenoid valves 107.

Movement of the synthesis plate 101 is performed by seating thesynthesis plate 101 on a movement stage 109 that can carry out preciseand repeatable movement patterns, controllable by a control device ormechanism (not shown). The control device may be, for example, amicrocontroller or other processor-based device (e.g., a laptop ortablet computer). The movement stage 109 can be, for example, an x-ystage, an R-θ stage, or other type of positioning system known in theart. Once the synthesis plate 101 has received the assigned reagents,and a pre-defined reaction time has occurred, the used reagents (nowreferred to as waste) are purged from the RIOS system 100. The waste ispurged by opening one or more solenoid valves 111 that are coupled toeach row of the synthesis plate 101 via one or more tubes 113. Anopening of the one or more solenoid valves 111 allows the inert gas inthe pressurized chamber to be purged to the outside environment, whichis at a lower pressure than the chamber 103. Since the only obstaclebetween the pressurized gas in the chamber 103 and an ambient pressureof the outside environment is the waste, the waste is carried out viathe one or more tubes 113 with the purged gas. Finally, the one or moresolenoid valves 111 are closed, an interior pressure of the chamber 103of the RIOS system 100 is restored, and the synthesis plate 101 (forexample, in a given row or well) is ready to undergo another reagentdelivery.

One feature of the RIOS system 100 described above is a drain block 119that couples the synthesis plate 101 to the one or more tubes 113. Theapparatus that accomplishes the purging operation described above withreference to row-independent oligonucleotide synthesis is now describedin more detail with reference to FIG. 2A-2D.

FIG. 2A shows an exemplary embodiment of a broken-out, disassembledapparatus 200 comprising a drain block 201, a sealing element 209, asynthesis plate 203, and a keeper 217 in accordance with variousembodiments of the disclosed subject matter. Consequently, FIG. 2A showsthe disassembled apparatus 200 for draining the synthesis plate 203 withreference to each of a set of rows 204 within the synthesis plate 203.As described below, the synthesis plate 203 may be any of a variety ofcommercially-available synthesis plates.

The drain block 201 is configured to accept, for example, the synthesisplate 203 in such a way that rows 204 in the synthesis plate 203 canundergo drainage via pressure, as described above, and in such a waythat these rows 204 can each experience a separate drainage withoutaffecting any adjacent or remaining ones of the rows 204. Each of therows contains a number of individual wells 221.

In a specific exemplary embodiment, the synthesis plate 203 comprises acommercially-available plate consisting of 384 wells (e.g., 384 of theindividual wells 221). As the 384-well plate is generally divided into16 rows of 24 wells each, the drain block 201, in this specificexemplary embodiment, is designed with a receiving feature 205 that has16 elongated openings 207 (one of the elongated openings 207 for each ofthe 16 rows in this example) for waste tips of each of the individualwells 221 to fall into. For example, in one specific exemplaryembodiment, given that the wells of all commercially-available 384-wellplates are spaced about 4.5 mm apart from center-to-center, theelongated openings 207 in the drain block 201 are cut to slightly overabout 108 mm long.

The bottom of the elongated opening 207 is dipped (e.g., a machined orotherwise formed depression) from about 20 mm to about 40 mm or more sothe waste may flow to a singular point: an opening leading to the backof the drain block 201. The dips of all the elongated openings 207 aredesigned in such a way that they do not conflict with each other. Inthis embodiment, the lowest point of one of the elongated openings 207is not the lowest point of another, using the lowest point as an outletso that the elongated openings 207 do not reach the same outlet.

In another embodiment, the outlets could be parallel to the elongatedopenings 207, thereby needing no variance in the lowest point as each ofthe elongated openings 207 since they would no longer run perpendicularto the outlets. Therefore, in any embodiment, the elongated openings 207of the receiving feature 205 of the drain block 201 remains anindependent vessel until a final termination at the back of the machine.

In a specific exemplary embodiment, the sealing element 209 of the drainblock 201 is a double-barreled or domed curve. The sealing element 209is, for example, a full-crown radius of about 2.54 m (approximately100.02 inches) applied to the receiving feature 205 after being machinedto an even height. The surrounding area is a recess 211 shaped toaccommodate the synthesis plate 203. The recess 211 is cut further thanthe sealing element 209 by about 2.54 mm (approximately 0.100 inches).These mechanical features of the drain block 201 allow for asubstantially even seal across the entire back of the synthesis plate203. Pressure from the synthesis plate 203, applied towards the centerof the receiving feature 205, with a gradual fall-off towards the outeredges of the receiving feature 205, allows for the gasket 213 to seatthe synthesis plate 203 substrate gap-free onto the drain block 201. Theradius stated above was determined in previous iterations of the drainblock 201 wherein no curvature was applied to the back of the synthesisplate 203. In that iteration, no seal was formed in the center of thesynthesis plate 203, though the edges experienced a slight resistance toalterations in surrounding pressures.

In an embodiment, the drain block 201 is machined out of Type-6061aluminum and type-2 hard-anodized to prevent against waste-causedcorrosion. However, a person of ordinary skill in the art, upon readingand understanding the disclosure provided herein, will recognize thatmaterials other than aluminum may be used. For example, the drain block201 may be machined from stainless steel or a number of other types ofmetallic or dielectric materials (e.g., aluminum oxide) depending on ause, cost, machining chars of the material, and other factors known to askilled artisan.

With continuing reference to FIG. 2A, and as shown, the gasket 213 isplaced between the synthesis plate 203 and the drain block 201. Thegasket 213 is cut with a set of holes 215, enough so that the, forexample, 384 waste tips (see e.g., waste tips 223 of FIG. 2D), locatedon the bottom of the synthesis plate 203, can fit into the384-complementary set of holes 215. In this embodiment, the tips 223 onthe back of the synthesis plate 203 are, for example, conical. Thelargest diameter on the waste tip measures slightly over about 2.54 mm(approximately 0.100″), therefore the set of holes 215 are cut to atolerance of about 0.127 mm (approximately 0.005″) less than 2.54 mm(approximately 0.100″) so that each individual hole of the set of holes215 may grip onto the waste tip 223. The gasket 213 is cut toaccommodate the full surface area available on the back of most plates,measuring about 110.7 mm (approximately 4.36 inches) long by about 73.4mm (approximately 2.89 inches) wide. The gasket 213 has a thickness ofabout 3.175 mm (approximately 0.125 inches). However, a person ofordinary skill in the art will recognize that a thickness of the gasket213 may be varied depending upon physical chars of the gasket 213, suchas a durometer of the material used to form the gasket 213.

In a specific exemplary embodiment, the material selected for the gasket213 was a 10A neoprene rubber (or another natural or synthetic rubber orsimilar flexible material) coated with a light film of grease. In onespecific exemplary embodiment, the grease used was a fluorocarbon-etherpolymer with the chemical formula F—(CF(CF₃)—CF₂—O)_(n)—CF₂CF₃. Thisgrease was chosen due to its inert properties and high load capacity. Adry, grease-less gasket may or may not provide a sufficient seal betweenthe rows 204 of the synthesis plate 203 above when coupled with thedrain block 201 beneath in various scenarios.

A keeper 217 is used to apply pressure onto the synthesis plate 203 andform a seal between the synthesis plate 203 and the drain block 201. Thedownward pressure applied to the synthesis plate 203 by the keeper 217is substantially even across all edges of the synthesis plate 203 sothat the sealing element 209 on the drain block 201 underneath may applyan opposing force to the center of the synthesis plate 203, and thatforce reaches across the synthesis plate 203 as the seal is formed fromthe center out. This coupling of the keeper 217, the synthesis plate203, the sealing element 209, and the drain block 201, allow a uniformseal to be achieved between all rows 204.

The top of the keeper 217 is machined or otherwise formed with a cut out219 that keeps each row 204 unobstructed so that each of the individualwells 221 in each of the rows 204 may receive incoming reagents.

FIG. 2B shows a top view of the synthesis plate 203 in such a way thateach of the individual wells 221 can be seen. FIG. 2C shows a side viewof the synthesis plate 203, and FIG. 2D shows an enlarged view of wastetips 223 of the synthesis plate 203.

FIG. 3 shows an exemplary embodiment of an assembled draining apparatus300 in accordance with various embodiments of the disclosed subjectmatter. Some of the parts shown may be similar or identical twocorresponding parts of FIG. 2A. In an exemplary embodiment, to pull akeeper 301 down onto a synthesis plate 311, a set of slots 303 for, forexample, 10-32 socket-head screws 305 have been cut into sides of thekeeper 301. A skilled artisan will recognize that many types ofmechanical fasteners or other types of chemical fasteners (e.g.,adhesives) may be used in addition to the socket-head screws 305 orinstead of the socket-head screws 305.

The socket-head screws 305 insert into respective ones of the set ofslots 303 and find their receiving thread in a shaft 307 that rotatesfreely inside of a drain block 309 beneath. Though originally designedwith six screw-positions (two on each end with two on opposite sides ofthe center), testing has found that as few as four screw-positions maybe used (e.g., the two on each end) with a 384-well plate. Additionally,degrees and evenness of pressure are largely applied to the synthesisplate 311 may even be superfluous once the grease (described above) isapplied. So long as the torque of each screw exceeds, for example,roughly about 2.71 N-m to about 4.07 N-m (approximately 2 ft-lbf toabout 3 ft-lbf), the seal holds at all ends of the synthesis plate 311.This being the case, a set of standard clips 313, toggle switches, orother mechanisms known in the art may provide just as effective a seal.

The assembled draining apparatus 300 shows that the keeper 301 may alsobe machined to accept a number of stand-offs 315 on the drain block 309.The stand-offs 315 provides at least two functions:

-   -   (1) To prevent or minimize over-tightening of the synthesis        plate 311 onto the drain block 309. The stand-offs 315 on the        drain block 309 are machined to about 4.78 mm (approximately        0.188 inches) above the top of the drain block 309 and receiving        holes 317 on the keeper 301 are machined so that the synthesis        plate 311 is not torqued over about 9.1 kg (approximately 20        lbs.). This limited torqueing is to prevent permanent warping of        the synthesis plate 311 (partially depending on a material from        which the synthesis plate 311 is formed); and    -   (2) To align the synthesis plate 311 properly onto the elongated        openings 207 (see FIG. 2A). When the stand-offs 315 and the        receiving holes 317 align, the synthesis plate 311 can be        seated. When the stand-offs 315 and the receiving holes 317 are        out of alignment, the synthesis plate 311 cannot be seated. This        alignment precaution is a failsafe against a user entering a run        of synthesis without waste tips of the synthesis plate 311        entering every elongated opening 207 (see FIG. 2A) of the drain        block 309.

Though commercially-available ones of the synthesis plates 311 that aredesigned for synthesis are not widely produced, the complete apparatushas been tested with products from two primary distributers of suchplates (e.g., Agilent Technologies, 5301 Stevens Creek Blvd, SantaClara, California, USA; and Biocomma Limited, Ground Floor, Bldg. 12,Zhonghaixin Innovative Industrial Park, Ganli Six Rd, Buji St, LonggangDistrict, Shenzhen, China) and has found success with each of theproducts of each company. Examples of such a plate is similar oridentical to the synthesis plate 311 of FIG. 3 and the synthesis plate203 of FIG. 2A-2C.

The assembled draining apparatus 300 is fully expandable so that, intheory, several configurations of multiple synthesis plates can beaccepted. FIG. 4 shows an exemplary embodiment of an expanded apparatus400 in accordance with various embodiments of the disclosed subjectmatter

Machining a drain block 401 with separate sets of sealing apparatusesand altering a set of waste outlets 411 to suit a number of availablewaste valves provides an effect similar to or the same as the drainblock 309 of FIG. 3 . However, the expanded apparatus 400 increases awork or production capacity by adding an additional synthesis plate 403.

To decide upon the appropriate drain configuration for the expandedapparatus 400, the number of waste valves needs to be considered. Then,a number of the additional synthesis plates 403 and a number of rows 405per synthesis plate 403 are counted so that the number of waste valvesis divided from the total number of the rows 405. This number determinesthe number of rows 405 that are to be drained by one valve. For example,if 16 valves were available to drain waste from two of the synthesisplates 403, with each of the synthesis plates 403 comprising 16 of therows 405 each (totaling 32 rows), the expanded apparatus 400 isconfigured to drain the two rows (in this case, the first of both of thesynthesis plates 403) simultaneously or substantially simultaneously, toachieve a purge equivalent or substantially equivalent to a singleversion of the synthesis plate 203, 311 system of FIGS. 2A and 3 . Insuch a configuration, the keeper 407 is accordingly expanded so that itmatches the length and width of an expanded version of the drain block401. A set of cut outs 409 similar or identical to the cut out of asingle version of the keeper 217 of FIG. 2A is cut so that each of thesynthesis plates 403 is given its own individual aperture for receivingdispensed reagents. Gaskets and sealing elements (not shown in FIG. 4 )remain the same or similar to the gaskets and sealing elements aspreviously described and be applied to each synthesis plate.

With concurrent reference again to FIG. 1 , in an exemplary embodiment,the expanded apparatus 400 allows for additional ones of the synthesisplates 403 to be added in a configuration that is complimentary to analignment of the dispensing tips located above the synthesis plates 101,on the ceiling 117 of the chamber 103. For example, if the same solventwas dispensed from tips aligned vertically, the synthesis plates 101 arealigned horizontally. This arrangement allows simultaneous firing of allaligned tips as the synthesis plates 101 move underneath. Thisarrangement also reduces or minimizes a need for movement in a verticaldirection, thus reducing an overall synthesis time.

FIG. 5 an exemplary embodiment of a drain-hole plate in accordance withvarious embodiments of the disclosed subject matter. With concurrentreference again to FIG. 2A, FIG. 5 is shown to include a single-welldrain block 500 that allows creation of an individual synthesis cycle oneach of the individual wells 221 on the synthesis plate 203. In thiscase, the elongated openings 207 in the existing format would instead bereplaced with a series of drain holes 501. Supplementary O-rings (notshown) incorporated in each of the drain holes 501 may be used to createa seal therein. In a similar way to how the number of waste outlets 411equals the number of rows 405 in FIG. 4 being drained substantiallysimultaneously, the single-well drain block 500 may use a monotonic(e.g., 1:1) relationship between the number of individual wells 221 inthe synthesis plate 203 and the number of waste outlets coupled to thesingle-well drain block 500. The drain holes 501 in the block may bemachined or otherwise formed so that their diameters (or othercharacteristic dimension) exceed the largest diameter (or othercharacteristic dimension) of the conical waste tip 223 (see FIG. 2D) onthe bottom of the synthesis plate 203 of FIG. 2A-2C. Accounting forthese alterations, the single-well drain block 500 can be used withinvarious embodiments described above (e.g., the RIOS) for autonomoussynthesis on each of the individual wells 221 of the synthesis plate203.

FIG. 6 an exemplary embodiment of a column-based drain block 600 inaccordance with various embodiments of the disclosed subject matter. Thecolumn-based drain block 600 facilitates existing columns ofcolumn-based synthesis. A block 601 can be machined or otherwise formedwith a series of holes 603 wide enough to use and seal typical synthesiscolumns with, for example, a friction fit (e.g., approximately 6 mm indiameter). The block 601 can be formed from any suitable materials suchas metals (e.g., aluminum or stainless steel) or dielectric materials(e.g., aluminum oxide or various types of plastics). The series of holes603 are, for example, drilled into a common channel 607 that flows to asingular output 609. The singular output 609 drains to a respectivewaste valve (not shown). The rest of the RIOS remains the same.

Regardless of the draining platform that facilitates the synthesis, thechemistry generally exists in an inert environment, oxygen free, andcontinues to receive and flush reagents without human intervention.Though the primary method for facilitating this process is described atthe beginning of this application, there are additional methods that canbe employed. Additional modifications to the various embodimentsdescribed above are described below.

FIG. 7 shows an exemplary embodiment of a gas/fluid pathway for a RIOSsystem under negative pressure in accordance with various embodiments ofthe disclosed subject matter. The chamber 701 in the RIOS system of FIG.7 could be constructed such that oxygen is not pushed out of, butactively substantially vacuumed from, the chamber 701 with a vacuum pump703. The negative pressure then created within the chamber 701 could benormalized with incoming gas from a pressurized gas-source 705 suitablefor oligonucleotide synthesis. The vacuum pump 703 used to pull gas fromthe chamber 701 directly would also pull waste from a synthesis plate707 through a drain block 709, removing used reagents (waste). Thevacuum pump 703 may be located downstream from a waste container 711 sothat the waste does not ever come into contact with the vacuum pump 703.The negative pressure created within the drain block 709 would bebrought back to the ambient pressure of the chamber 701 so as not toallow fresh reagents to fall through the synthesis plate 707 unused.This condition allows drain lines protruding from the drain block 709 toreach an array of corresponding valves 713 where the vacuum pump 703 islocated downstream of the array of corresponding valves 713. In anembodiment, the vacuum pump 703 may be accessed individually by eachvalve via a manifold (not shown) to which all of the array ofcorresponding valves 713 connect. Normalizing a pressure of the manifoldallows for a positive pressure to selectively be distributed back to thedrain block 709 so that any recently drained row of the synthesis plate707 could be brought to a current ambient pressure of the chamber 701.

FIG. 8 shows an exemplary embodiment of a gas/fluid pathway for a RIOSsystem under a hybridization of positive pressure and negative pressurein accordance with various embodiments of the disclosed subject matter.FIG. 8 therefore shows a combined approach wherein a synthesis chamber801 is substantially vacuumed of oxygen (or other gas) with a vacuumpump 803 while a synthesis plate 805 experiences positive pressure froman incoming, pressurized gas-source 807. The pressurized gas-source 807purges waste from the synthesis plate 805 into a waste container 809.Once the synthesis chamber 801 has been vacuumed of one or morecorrosive gases and has been pressurized to a desired level of inertgas, the remainder of the RIOS system could remain consistent with otherembodiments disclosed herein.

Additional mechanisms designed for an improvement of optimization offluidic handling are submitted and detailed below.

For example, FIG. 9 shows an exemplary embodiment of a dispense-tipassembly 900 in accordance with various embodiments of the disclosedsubject matter. For a dispensing one or more chemicals from one of theset of solenoid valves 107 (see FIG. 1 ) to insert into a chamber 103without a loss of pressure, flanged fittings 901 have been designed toaccept common fluidic lines, for example, having an outside diameter(OD) of about 3.175 mm (approximately 0.125 inches). In a specificexemplary embodiment, various fluidic lines can be fitted with thedispense-tip assembly 900, which can direct the line so as not to allowthe fluidic line to dispense in unwanted directions. The dispense-tipassembly 900 may also contain, for example, an imbedded O-ring groove903 to seal pressure within the chamber and be machined with a flatsurface 905 on the receiving end of a dispense nozzle 908 to allow theflanged fitting 901 to press against the flat surface 905 to prevent orreduce leaks along the fluidic path (such fittings are available from,for example, IDEX Corporation, 1925 West Field Court, Suite 200, LakeForest, Illinois, USA; or Valco Instruments Company Inc., 8300Waterbury, Houston, Texas, USA).

The flat surface 905 of the dispense-tip assembly 900 has a diameterwide enough to accept, for example, ¼′-28 (or a substantiallymetric-equivalent) flanged fittings 901. A union 909 connects thereceiving end of the dispense nozzle 908 to the flanged fitting with acomplementary thread 913. Though the dispense-tip assembly 900 could bedesigned to accept a flange of any diameter, the ¼″-28 flanged fittings901 were chosen due to their flexibility in receiving fluidic lines ofvarying ODs.

A through-hole 911 in the center of the dispense-tip assembly 900 ismachined to the OD of the incoming line, in this case, about 3.175 mm(approximately 0.125 inch). No reduction in diameter is needed at anypoint along the through-hole 911. The flanged fitting 901 seals pressureagainst the flat surface 905 of the nozzle and allows the remainder ofthe line to come through the dispense nozzle 908 while leaving apressure of the chamber 103 (see FIG. 1 ) pressure constant.

In embodiments, the union 909 may also act as a tightening agent thatpulls the dispensing portion of the dispense nozzle 908 (located, forexample, inside the chamber 103 of FIG. 1 ) against the ceiling 117(also of FIG. 1 ) from which it protrudes. In this embodiment, theportion of the dispense-tip assembly 900 that snugs against the ceiling117 may be machined wider than the hole in the ceiling 117. In thisexample, this wider portion may be machined to seat a small O-ring ofabout 6.35 mm ID by about 0.792 mm W (approximately 0.250 inchID×approximately 0.0312 inch W) along its edges. Additionally, a smallamount of grease can be applied to this O-ring to improve the seal evenfurther. When dispense-tip assembly 900 is utilized, the reagents candispense without a need to adjust line direction and without a concernof leaking gas from the chamber 103.

FIG. 10 shows an exemplary embodiment of a lid housing 1000 forvalve-adjacent reagent bottles in accordance with various embodiments ofthe disclosed subject matter. The lid housing 1000 of FIG. 10 maytherefore be considered an alternative embodiment of the ceiling 117 ofFIG. 1 . The lid housing 1000 used to seal the chamber 103 of FIG. 1 (orany of the various other embodiments) is configured to perform multiplefunctions. For example, the lid housing 1000 serves to seal inert gasinside the chamber 103. The lid housing 1000 is also hinged to allowusers access into the chamber 103 for the insertion and collection ofsynthesis plates. Unlike the ceiling 117 of FIG. 1 , the lid housing1000 of FIG. 10 includes a cavity 1001 to house the bottles 1007 for thesynthesis process. The lid housing 1000 further includes an opening 1003to grant access to a valve array 1005. The valve array 1005 allowsdispensing into, for example, each of the individual wells 115 of thesynthesis plate 101 (see FIG. 1 ).

As most amidites used to construct genetic material during synthesis canhave high costs, ranging into the thousands of dollars per gram,restricted line lengths and minimal dead volumes may be a significantconcern to an end user. Not only are wasted amidites costly to replace,their expiration inside fluidic lines can result in crystallization,leading to an inefficient or blocked dispense. Because the movement ofthe fluid within the line is generally one-way, the full contents of theline are utilized, sometimes unnecessarily, in order to avoid wastedamidites during and/or after synthesis.

To resolve issues regarding line length, the lid housing 1000 thatassist in directing the valve array 1005 into the individual wells 115of the synthesis plate 101 can be fitted to allow a rack to hold thebottles 1007 be formed on one or more sides of the lid housing 1000. Thematerial (e.g., sheet metal or other suitable material) selected to holdthe bottles 1007 in this way may contain, for example, two separatewalls (e.g., steel wall dividers) that keep the bottles 1007 frominteracting with any of the valves within the valve array 1005, therebyconstituting a safety measure to protect the end user should a leakoccur. A bottle receptacle 1011 can be used on the rack for the bottles1007, into which the bottles fit. The bottle receptacle 1011 may push anO-ring or other sealing device against the opening of the bottle so thatfluid does not leak from the bottle 1007 when the lid housing 1000 israised. The O-ring or other sealing device also seals gas within thebottle so that a positive pressure used to displace, for example, theliquid amidite is preserved.

With reference now to FIG. 11 , an exemplary embodiment of areverse-flush pathway 1100 in accordance with various embodiments of thedisclosed subject matter is shown. As an additional feature to performfluid handling, various embodiments of the RIOS system may include anexhaust valve 1101 that is fitted downstream from a pressure source1103, but before entry into a set of bottles 1105 and an array ofsolenoid valves 1107. By pressurizing a chamber 1109, to which fluidiclines 1111 lead, and relieving pressure from the set of bottles 1105from which the fluids originated, existing gas in the chamber 1109 canbe used to push excess reagents back to the bottles 1105. The gas flowsfrom the chamber 1109, through the fluidic lines 1111, pushing theliquid into the bottles 1105, and creating a volume with respective onesof the bottles 1105 by way of the exhaust valve 1101 relieving pressurefrom the bottles 1105. The pressure differential between the chamber1109 and the bottles 1105 creates a reverse-flush embodiment for theuser that would further decrease wasted reagents and/or amidites at theend of each synthesis.

FIG. 12 shows an exemplary embodiment of a calibration apparatus 1200that may be used with various ones of the embodiments disclosed herein.The calibration apparatus 1200 is designed to calibrate various ones ofthe solenoid valves disclosed herein (e.g., the solenoid valves 107 ofFIG. 1 ).

The exemplary embodiment of FIG. 12 is shown to include a plate 1203(e.g., comprising metal or other suitable material) formed with a seriesof holes 1205 to seat standard tubules 1207 used in polymerase chainreaction (PCR) devices. The series of holes 1205 is formed in sets of,for example, three, with each one of the series of holes 1205 receivingits own tubule 1207 (e.g., a PCR tubule). The tubules 1207 (one per hole1205 in the set) are used to measure liquids dispensed by a solenoidvalve in, for example, three separate increments, such as 50millisecond, 100 millisecond, and 200 millisecond increments. Contentsof the tubule 1207 is now the “dispensed liquid.” A weight or volume ofthe dispensed liquid is measured, an average is taken, with a density ofthe dispensed liquid being considered, and the result gives an accuratetime-to-liquid delivery value.

By employing a simple latching device 1209 that is coupled to, forexample, the drain block 201 of FIG. 2A in the same or similar way tohow the keeper 217 is coupled to the drain block 201, the calibrationapparatus 1200 can be used to calibrate dispensing valves as describedabove with regard to any of the preceding exemplary embodiments. Uponreading and understanding the disclosed subject matter, a person ofordinary skill in the art will recognize that any amount of dispenses,holes 1205, and tubules 1207 can be used, although increasing thenumbers will result in a more robust average. Similarly, dispense timesare used as examples only.

Using the unique apparatus along with the additional design elements andmethods described, the end user is left with a machine far superior tothose existing in today's oligonucleotide synthesis market. The machineallows plate-based synthesis to compete with conventional “column-based”synthesis by shortening cycle times and reducing waste, while creatingsmaller quantities of oligonucleotides in higher yields and widervarieties at no additional cost.

THE FOLLOWING NUMBERED EXAMPLES ARE EMBODIMENTS OF THE DISCLOSED SUBJECTMATTER

Example 1: In an embodiment, the disclosed subject matter includes apressurized system designed to facilitate the synthesis ofoligonucleotides on a synthesis plate with respect to rows using apositive-pressure system, a row-independent oligonucleotide synthesis(RIOS) system.

Example 2: In an embodiment, the disclosed subject matter includes anapparatus used in oligonucleotide synthesis. The apparatus includes amachined block configured to receive a commercially-available synthesisplate, a keeper to apply pressure to the commercially-availablesynthesis plate, and a sealing element to seal thecommercially-available synthesis plate to the machined block.

Example 3: A modified apparatus of either of the two preceding Examples,wherein the keeper and the machined block are configured to belengthened so as to allow the addition of one or more synthesis plates.

Example 4: The modified apparatus of any one of the preceding examples,wherein the drain block is configured to accept commercially-availablesynthesis plates and drain each well of the plate individually.

Example 5: The modified apparatus of any one of the preceding examples,wherein the drain block is configured to perform synthesis withcommercially-available synthesis columns.

Example 6: In various embodiments, the disclosed subject matter includesan apparatus used in oligonucleotide synthesis. The apparatus includes achamber configured to facilitate the synthesis of the chemistry withinthe apparatus via a selection of pressures including a positive pressureand a negative pressure. Example 7: In various embodiments, thedisclosed subject matter includes an apparatus used in oligonucleotidesynthesis. The apparatus includes a chamber configured to facilitate thesynthesis of chemistry within the apparatus via a selection of pressuresincluding a hybridization of both positive pressure and negativepressure.

Example 8: An apparatus of any one of the preceding examples, furthercomprising machined dispense-tips to be coupled in close proximity tovalves installed on a lid of a pressurized chamber, the machineddispense-tips being spaced and aligned to be substantially matched torespective distances of the commercially-available synthesis plate. Themachined dispense-tips include a support for a fluidic line, the supportcomprising at least one of a flange or a ferrule and O-ring.

Example 9: An apparatus of any one of the preceding examples, furthercomprising a lid sealing the chamber having both bottles and valvesproximately coupled to reduce dead volume.

Example 10: An apparatus of any one of the preceding examples, furthercomprising a valve-to-manifold mechanism to flush at least a portion ofresidual reagents that accumulate in the fluidic lines post-synthesisback to their respective points-of-origin.

Example 11: An apparatus of any one of the preceding examples, furthercomprising a machined plate designed to fit and latch onto the existingdrain for calibration of solenoid valves in a RIOS system.

Throughout this specification, plural instances may implementcomponents, operations, or structures described as a single instance.Although individual operations of one or more methods are illustratedand described as separate operations, one or more of the individualoperations may be performed concurrently, and nothing requires that theoperations be performed in the order illustrated. Structures andfunctionality presented as separate components in example configurationsmay be implemented as a combined structure or component. Similarly,structures and functionality presented as a single component may beimplemented as separate components. These and other variations,modifications, additions, and improvements fall within the scope of thesubject matter herein.

As used herein, the term “or” may be construed in an inclusive orexclusive sense. Further, other embodiments will be understood by aperson of ordinary skill in the art upon reading and understanding thedisclosure provided. Further, upon reading and understanding thedisclosure provided herein, the person of ordinary skill in the art willreadily understand that various combinations of the techniques andexamples provided herein may all be applied in various combinations.

Although various embodiments are discussed separately, these separateembodiments are not intended to be considered as independent techniquesor designs. As indicated above, each of the various portions may beinter-related and each may be used separately or in combination withother embodiments. For example, although various embodiments of methods,operations, and processes have been described, these methods,operations, and processes may be used either separately or in variouscombinations.

Consequently, many modifications and variations can be made, as will beapparent to a person of ordinary skill in the art upon reading andunderstanding the disclosure provided herein. Functionally equivalentmethods and devices within the scope of the disclosure, in addition tothose enumerated herein, will be apparent to the skilled artisan fromthe foregoing descriptions. Portions and features of some embodimentsmay be included in, or substituted for, those of others. Suchmodifications and variations are intended to fall within a scope of theappended claims. Therefore, the present disclosure is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

The Abstract of the Disclosure is provided to allow the reader toquickly ascertain the nature of the technical disclosure. The abstractis submitted with the understanding that it will not be used tointerpret or limit the claims. In addition, in the foregoing DetailedDescription, it may be seen that various features may be groupedtogether in a single embodiment for the purpose of streamlining thedisclosure. This method of disclosure is not to be interpreted aslimiting the claims. Thus, the following claims are hereby incorporatedinto the Detailed Description, with each claim standing on its own as aseparate embodiment.

What is claimed is:
 1. An apparatus comprising: a chamber, the chamberunder an interior pressure; two or more synthesis plates inside thechamber, each of the two or more synthesis plates having a plurality ofrows, each row of the plurality of rows comprising a plurality of wellsand each well of the plurality of wells comprising a waste tip on abottom of the each of the two or more synthesis plates; a drain blocklocated beneath the two or more synthesis plates; a keeper located ontop of the two or more synthesis plates, the keeper fastened to thedrain block by mechanical fasteners, the mechanical fasteners applyingpressure to the two or more synthesis plates, thereby forming a sealaround each of the plurality of rows and each of the plurality of wellsof the two or more synthesis plates; a gas source operably coupled tothe chamber via one or more tubes; and a plurality of solenoid valves,the plurality of solenoid valves dispensing reagents to the plurality ofwells of the two or more synthesis plates.
 2. The apparatus of claim 1,further comprising a movement stage, the movement stage moving the twoor more synthesis plates under the plurality of solenoid valves.
 3. Theapparatus of claim 1, where the interior pressure is a positivepressure.
 4. The apparatus of claim 1, further comprising a plurality ofwaste solenoid valves, each of the plurality of waste solenoid valvesoperably coupled to one or more rows of the two or more synthesisplates.
 5. The apparatus of claim 1, further comprising a vacuum pumpoperably connected to the chamber via one or more tubes.
 6. Theapparatus of claim 5, where the interior pressure is a negativepressure.
 7. The apparatus of claim 5, where the interior pressure is ahybridization of a positive pressure and a negative pressure.
 8. Theapparatus of claim 1, further comprising a dispense-tip assembly.
 9. Anapparatus comprising: a chamber, the chamber under an interior pressure;two or more synthesis plates inside the chamber, each of the two or moresynthesis plates having a plurality of rows, each row of the pluralityof rows comprising a plurality of wells and each well of the pluralityof wells comprising a waste tip on a bottom of the each of the two ormore synthesis plates; a drain block located beneath the two or moresynthesis plates; a keeper located on top of the two or more synthesisplates, the keeper fastened to the drain block by mechanical fasteners,the mechanical fasteners applying pressure to the two or more synthesisplates, thereby forming a seal around each of the plurality of rows andeach of the plurality of wells of the two or more synthesis plates; agas source operably coupled to the chamber via one or more tubes; and aplurality of solenoid valves, the plurality of solenoid valvesdispensing reagents through a ceiling of the chamber to the plurality ofwells of the two or more synthesis plates.
 10. The apparatus of claim 9,further comprising a plurality of waste solenoid valves, each of theplurality of waste solenoid valves operably coupled to one or more rowsof the two or more synthesis plates.
 11. The apparatus of claim 9,wherein the interior pressure is a positive pressure.
 12. The apparatusof claim 9, further comprising a vacuum pump operably connected to thechamber via one or more tubes.
 13. The apparatus of claim 12, whereinthe interior pressure is a negative pressure.
 14. The apparatus of claim12, wherein the interior pressure is a hybridization of a positivepressure and a negative pressure.
 15. The apparatus of claim 9, furthercomprising further comprising a dispense-tip assembly, including adispense nozzle.
 16. The apparatus of claim 15, where the dispensenozzle is inside of the chamber against the ceiling.
 17. An apparatuscomprising: a chamber comprising a lid; two or more synthesis platesinside the chamber, each of the two or more synthesis plates having aplurality of rows, each row of the plurality of rows comprising aplurality of wells and each well of the plurality of wells comprising awaste tip on a bottom of the each of the two or more synthesis plates; adrain block located beneath the two or more synthesis plates; a keeperlocated on top of the two or more synthesis plates, the keeper fastenedto the drain block by mechanical fasteners, the mechanical fastenersapplying pressure to the two or more synthesis plates, thereby forming aseal around each of the plurality of rows and each of the plurality ofwells of the two or more synthesis plates; and a gas source operablycoupled to the chamber via one or more tubes, the gas source creating apressure inside the chamber.
 18. The apparatus of claim 17, where thelid comprises a cavity with a plurality of bottles containing reagentsinside the cavity.
 19. The apparatus of claim 18, further comprising anopening in the lid, the opening containing a valve array, the valvearray dispensing the reagents from the plurality of bottles to theplurality of wells of the two or more synthesis plates.
 20. Theapparatus of claim 17, further comprising a plurality of waste solenoidvalves, each of the plurality of waste solenoid valves operably coupledto one or more rows of the two or more synthesis plates.